PROJECT SUMMARY Triple-negative breast cancer (TNBC) is an aggressive subtype in which patients display extensive intratumor genomic heterogeneity and frequently (50%) develop resistance to neoadjuvant chemotherapy (NAC) which leads to metastatic disease and death. Due to the absence of hormonal receptors and targeted therapies, TNBC patients with refractory disease are often left with limited treatment options. Currently, our understanding of the genomic evolution of tumor cells and the role of the tumor microenvironment in chemoresistant disease at primary and metastatic organ sites represents a major gap in knowledge that this proposal aims to address. Our group has developed cutting-edge single cell DNA and RNA sequencing technologies that can overcome previous technical barriers and limitations with `bulk' genomic methods for studying the genomic and phenotypic evolution of tumor cells in response to chemotherapy. Our preliminary data in a small number of TNBC patients suggests that genomic evolution of chemoresistance occurs through the adaptive selection of pre-existing mutations and copy number alterations, which is followed by transcriptional reprogramming, to achieve a chemoresistant phenotype (Kim et al. 2018, Cell). We further hypothesize that transcriptional reprogramming of cell types in the tumor microenvironment occurs in chemoresistant disease and that resistance programs are clonally inherited at distant metastatic organ sites. To comprehensively investigate these questions in matched longitudinal samples from TNBC patients in a large neoadjuvant chemotherapy trial (ARTEMIS), we propose three synergistic aims: Aim 1 will determine if copy number aberrations (CNAs) and subclonal mutations associated with chemoresistance are pre-existing and adaptively selected in response to therapy. Aim 2 will investigate if tumor cells and cell types in the microenvironment undergo transcriptional reprogramming in refractory disease. Aim 3 will determine if subpopulations of resistant cells in the primary tumor seed the metastatic lesions and confer resistance programs at distant organ sites. Completion of these aims will define the genomic and evolutionary basis of chemoresistance in TNBC patients and will provide new diagnostic biomarkers and therapeutic targets for overcoming chemoresistant disease, which is a critical unmet clinical need. Our long-term goal is to translate single cell sequencing technologies into the clinic, where they are poised to have a major impact on the diagnosis and treatment of breast cancer patients. The proposed aims are directly aligned with the mission of NIH to reduce morbidity and improve the quality of life for breast cancer patients.